Effect of p95HER2/611CTF on the response to trastuzumab and chemotherapy.

Human epidermal growth factor receptor 2 (HER2)-positive breast cancers are currently treated with trastuzumab, an anti-HER2 antibody. About 30% of these tumors express a group of HER2 fragments collectively known as p95HER2. Our previous work indicated that p95HER2-positive tumors are resistant to trastuzumab monotherapy. However, recent results showed that tumors expressing the most active of these fragments, p95HER2/611CTF, respond to trastuzumab plus chemotherapy. To clarify this discrepancy, we analyzed the response to chemotherapy of cell lines transfected with p95HER2/611CTF and patient-derived xenografts (n = 7 mice per group) with different levels of the fragment. All statistical tests were two-sided. p95HER2/611CTF-negative and positive tumors showed different responses to various chemotherapeutic agents, which are particularly effective on p95HER2/611CTF-positive cells. Furthermore, chemotherapy sensitizes p95HER2/611CTF-positive patient-derived xenograft tumors to trastuzumab (mean tumor volume, trastuzumab alone: 906 mm(3), 95% confidence interval = 1274 to 538 mm(3); trastuzumab+doxorubicin: 259 mm(3), 95% confidence interval = 387 to 131 mm(3); P < .001). This sensitization may be related to HER2 stabilization induced by chemotherapy in p95HER2/611CTF-positive cells.

Constitutive HER2 signaling promotes breast cancer metastasis through cellular senescence.

Senescence, a terminal cell proliferation arrest, can be triggered by oncogenes. Oncogene-induced senescence is classically considered a tumor defense barrier. However, several findings show that, under certain circumstances, senescent cells may favor tumor progression because of their secretory phenotype. Here, we show that the expression in different breast epithelial cell lines of p95HER2, a constitutively active fragment of the tyrosine kinase receptor HER2, results in either increased proliferation or senescence. In senescent cells, p95HER2 elicits a secretome enriched in proteases, cytokines, and growth factors. This secretory phenotype is not a mere consequence of the senescence status and requires continuous HER2 signaling to be maintained. Underscoring the functional relevance of the p95HER2-induced senescence secretome, we show that p95HER2-induced senescent cells promote metastasis in vivo in a non-cell-autonomous manner.

p95HER2 and breast cancer.

A subtype of HER2-positive tumors with distinct biological and clinical features expresses a series of carboxy-terminal fragments collectively known as p95HER2. One of these fragments, named 100- to 115-kDa p95HER2 or 611-CTF, is hyperactive because of its ability to form homodimers maintained by intermolecular disulfide bonds. Despite lacking the majority of the extracellular domain, this HER2 fragment drives breast cancer progression in vivo. The recent availability of specific anti-p95 antibodies has confirmed previous results indicating that the expression of p95HER2 is predictive of poor prognosis and correlates with resistance to the treatment with trastuzumab, a therapeutic antibody directed against the extracellular domain of HER2.

A major role of p95/611-CTF, a carboxy-terminal fragment of HER2, in the down-modulation of the estrogen receptor in HER2-positive breast cancers.

Current classification of breast cancers depends in great part on the expression of human epidermal growth factor receptor 2 (HER2), a cell surface tyrosine kinase receptor, and estrogen receptor (ER), the nuclear receptor for estrogen. In addition to reliable biomarkers, these receptors are targets of effective and widely used antitumor drugs. During malignant progression, HER2 and ER can establish an intricate cross-talk. In some cases, HER2 overexpression leads to the downregulation of ER and undermining of anti-ER therapies. A subgroup of HER2-positive breast cancer patients with poor prognosis expresses a heterogeneous collection of HER2 carboxy-terminal fragments (CTF) collectively known as p95HER2. One of these fragments, 611-CTF, is oncogenic in a variety of preclinical models. However, because of the lack of an appropriate tool to specifically analyze its levels in the clinical setting, the value of 611-CTF as a biomarker has not been established yet. Here, we show that 611-CTF induces resistance to antiestrogen therapy and a more pronounced down-modulation of ER than that induced by full-length HER2. To validate this effect in breast cancer samples, we developed specific anti-611-CTF antibodies. With these antibodies, we showed that, whereas the frequency of ER positivity in HER2-positive/611-CTF-negative tumors (72.6%) is similar to that reported for HER2-negative tumors (70-80%), the number of ER-positive tumors in the 611-CTF-positive subgroup is very low (31.2%). These results reveal a mechanism of ER regulation mediated by HER2, which suggests a new strategy to improve responses to endocrine therapy in breast cancer.

HER2 fragmentation and breast cancer stratification.

HER2 is a tyrosine kinase receptor whose overexpression in breast cancers correlates with poor prognosis. A subset of HER2-positive tumors also expresses a series of HER2 fragments, collectively known as p95HER2. These fragments are emerging as a valuable biomarker for this subset of patients, who have a particularly poor prognosis.

HER2 carboxyl-terminal fragments regulate cell migration and cortactin phosphorylation.

A group of breast cancer patients with a higher probability of developing metastasis expresses a series of carboxyl-terminal fragments (CTFs) of the tyrosine kinase receptor HER2. One of these fragments, 611-CTF, is a hyperactive form of HER2 that constitutively establishes homodimers maintained by disulfide bonds, making it an excellent model to study overactivation of HER2 during tumor progression and metastasis. Here we show that expression of 611-CTF increases cell motility in a variety of assays. Since cell motility is frequently regulated by phosphorylation/dephosphorylation, we looked for phosphoproteins mediating the effect of 611-CTF using two alternative proteomic approaches, stable isotope labeling with amino acids in cell culture and difference gel electrophoresis, and found that the latter is particularly well suited to detect changes in multiphosphorylated proteins. The difference gel electrophoresis screening identified cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, as a phosphoprotein probably regulated by 611-CTF. This result was validated by characterizing cortactin in cells expressing this HER2 fragment. Finally, we showed that the knockdown of cortactin impairs 611-CTF-induced cell migration. These results suggest that cortactin is a target of 611-CTF involved in the regulation of cell migration and, thus, in the metastatic behavior of breast tumors expressing this CTF.

A naturally occurring HER2 carboxy-terminal fragment promotes mammary tumor growth and metastasis.

HER2 is a tyrosine kinase receptor causally involved in cancer. A subgroup of breast cancer patients with particularly poor clinical outcomes expresses a heterogeneous collection of HER2 carboxy-terminal fragments (CTFs). However, since the CTFs lack the extracellular domain that drives dimerization and subsequent activation of full-length HER2, they are in principle expected to be inactive. Here we show that at low expression levels one of these fragments, 611-CTF, activated multiple signaling pathways because of its unanticipated ability to constitutively homodimerize. A transcriptomic analysis revealed that 611-CTF specifically controlled the expression of genes that we found to be correlated with poor prognosis in breast cancer. Among the 611-CTF-regulated genes were several that have previously been linked to metastasis, including those for MET, EPHA2, matrix metalloproteinase 1, interleukin 11, angiopoietin-like 4, and different integrins. It is thought that transgenic mice overexpressing HER2 in the mammary glands develop tumors only after acquisition of activating mutations in the transgene. In contrast, we show that expression of 611-CTF led to development of aggressive and invasive mammary tumors without the need for mutations. These results demonstrate that 611-CTF is a potent oncogene capable of promoting mammary tumor progression and metastasis.

Features in the N and C termini of the MAPK-interacting kinase Mnk1 mediate its nucleocytoplasmic shuttling.

Eukaryotic initiation factor eIF4E binds to the 5'-cap structure of the mRNA and also to the molecular scaffold protein eIF4G. eIF4E is a phosphoprotein, and the kinases that act on it have been identified as the MAPK-interacting kinases Mnk1 and Mnk2. Mnk1/2 also bind to the scaffold protein eIF4G. The N-terminal region of Mnk1 has previously been shown to bind to importin alpha, a component of the nuclear transport machinery, although Mnk1 itself is cytoplasmic. Here we identify a CRM1-type nuclear export motif in the C-terminal part of Mnk1. Substitution of hydrophobic residues in this motif results in Mnk1 becoming nuclear. This has allowed us to study the features of Mnk1 that are involved in its transport to the nucleus. This process requires part, but not all, of a polybasic region near the N terminus of Mnk1. Residues required for nuclear transport are also required for its interaction with importin alpha. This polybasic region also serves a second function in that it is required for the binding of Mnk1 to eIF4G, although the residues involved in this interaction are not identical to those involved in the binding of Mnk1 to importin alpha. Interaction of Mnk1 with eIF4G promotes the phosphorylation of eIF4E. Mutations that reduce the binding of Mnk1 to eIF4G in vivo and in vitro also decrease the ability of Mnk1 to enhance eIF4E phosphorylation in vivo, underlining the importance of the eIF4G-Mnk1 interaction in this process.